Neuroendocrine characterization and anorexigenic effects of telmisartan in diet- and glitazone-induced weight gain.

Telmisartan is an angiotensin II receptor blocker with peroxisome proliferator-activated receptor-gamma agonistic properties. Telmisartan prevents weight gain and decreases food intake in models of obesity and in glitazone-treated rodents. This study further investigates the influence of telmisartan and pioglitazone and their association on weight gain and body composition by examining their influence on neuroendocrine mediators involved in food intake. Male C57/Black 6 mice were fed a high-fat diet, weight matched, and randomized in 4 treatment groups: vehicle, pioglitazone, telmisartan, and pioglitazone-telmisartan. Weight gain, food and water intake, body composition, plasma leptin levels, and the hypothalamic expression of neuroendocrine mediators were analyzed. Additional studies were performed with irbesartan and in angiotensin II 1(A) receptor-knockout mice. Telmisartan abolished weight and fat gain in vehicle- and pioglitazone-treated mice while decreasing food intake, the hypothalamic expression of the agouti-related protein, and plasma leptin levels. Modifications in neuropeptide Y and proopiomelanocortin were not consistent with changes in food intake. The effects on weight gain and expression of the agouti-related protein were intermediate with irbesartan. The effects of telmisartan on weight gain were even more pronounced in angiotensin II 1(A) receptor-knockout mice. This study confirms the anorexigenic effects of telmisartan in mice fed a high-fat diet and suggests for the first time a functional role of telmisartan on hypothalamic orexigenic agouti-related protein regulation. These anorexigenic properties abolish both weight gain and body composition modifications in fat-fed and glitazone-treated mice. The anorexigenic properties are independent from the angiotensin II 1(A) receptor.

Unusual astrocyte reactivity caused by the food mycotoxin ochratoxin A in aggregating rat brain cell cultures.

Ochratoxin A (OTA), a mycotoxin and widespread food contaminant, is known for its patent nephrotoxicity and potential neurotoxicity. Previous observations in vitro showed that in the CNS, glial cells were particularly sensitive to OTA. In the search for the molecular mechanisms underlying OTA neurotoxicity, we investigated the relationship between OTA toxicity and glial reactivity, in serum-free aggregating brain cell cultures. Using quantitative reverse transcriptase-polymerase chain reaction to analyze changes in gene expression, we found that in astrocytes, non cytotoxic concentrations of OTA down-regulated glial fibrillary acidic protein, while it up-regulated vimentin and the peroxisome proliferator-activated receptor-gamma expression. OTA also up-regulated the inducible nitric oxide synthase and the heme oxygenase-1. These OTA-induced alterations in gene expression were more pronounced in cultures at an advanced stage of maturation. The natural peroxisome proliferator-activated receptor-gamma ligand, 15-deoxy-delta(12,14) prostaglandin J2, and the cyclic AMP analog, bromo cyclic AMP, significantly attenuated the strong induction of peroxisome proliferator-activated receptor-gamma and inducible nitric oxide synthase, while they partially reversed the inhibitory effect of OTA on glial fibrillary acidic protein. The present results show that OTA affects the cytoskeletal integrity of astrocytes as well as the expression of genes pertaining to the brain inflammatory response system, and suggest that a relationship exists between the inflammatory events and the cytoskeletal changes induced by OTA. Furthermore, these results suggest that, by inducing an atypical glial reactivity, OTA may severely affect the neuroprotective capacity of glial cells.

Do peroxisome proliferating compounds pose a hepatocarcinogenic hazard to humans?

The purpose of the workshop "Do Peroxisome Proliferating Compounds Pose a Hepatocarcinogenic Hazard to Humans?" was to provide a review of the current state of the science on the relationship between peroxisome proliferation and hepatocarcinogenesis. There has been much debate regarding the mechanism by which peroxisome proliferators may induce liver tumors in rats and mice and whether these events occur in humans. A primary goal of the workshop was to determine where consensus might be reached regarding the interpretation of these data relative to the assessment of potential human risks. A core set of biochemical and cellular events has been identified in the rodent strains that are susceptible to the hepatocarcinogenic effects of peroxisome proliferators, including peroxisome proliferation, increases in fatty acyl-CoA oxidase levels, microsomal fatty acid oxidation, excess production of hydrogen peroxide, increases in rates of cell proliferation, and expression and activation of the alpha subtype of the peroxisome proliferator-activated receptor (PPAR-alpha). Such effects have not been identified clinically in liver biopsies from humans exposed to peroxisome proliferators or in in vitro studies with human hepatocytes, although PPAR-alpha is expressed at a very low level in human liver. Consensus was reached regarding the significant intermediary roles of cell proliferation and PPAR-alpha receptor expression and activation in tumor formation. Information considered necessary for characterizing a compound as a peroxisome proliferating hepatocarcinogen include hepatomegaly, enhanced cell proliferation, and an increase in hepatic acyl-CoA oxidase and/or palmitoyl-CoA oxidation levels. Given the lack of genotoxic potential of most peroxisome proliferating agents, and since humans appear likely to be refractive or insensitive to the tumorigenic response, risk assessments based on tumor data may not be appropriate. However, nontumor data on intermediate endpoints would provide appropriate toxicological endpoints to determine a point of departure such as the LED10 or NOAEL which would be the basis for a margin-of-exposure (MOE) risk assessment approach. Pertinent factors to be considered in the MOE evaluation would include the slope of the dose-response curve at the point of departure, the background exposure levels, and variability in the human response.

Changes in gene expression profile in human subcutaneous adipose tissue during significant weight loss.

Objective: To analyze the expression of peroxisome proliferator-activated receptor-γ1 and 2 (PPARγ1 and 2), 11β-hydroxysteroid dehydrogenase type 1 (11βHSD1), and leptin in adipose tissue (AT) of obese women during weight loss following Roux-en-Y gastric bypass (RYGB) and to compare these levels with those obtained in AT of nonobese subjects. Methods: Gene expression was determined by real-time RT-PCR prior to surgery and at 3, 6, and 12 months after RYGB. Results: All obese patients lost weight, reaching a mean BMI of 29.3 ± 1.0 kg/m(2) at 1 year after surgery (-33.9 ± 1.5% of their initial body weight). In obese subjects leptin and 11βHSD1 were over-expressed, whereas PPARγ1 was expressed at lower levels compared to controls. After surgery, leptin and 11βHSD1 gene expression decreased, whereas PPARγ1 expression increased. At 12 months after RYGB, these 3 genes had reached levels similar to the controls. In contrast, PPARγ2 gene expression was not different between groups and types of tissue and remained unchanged during weight loss. We found a positive correlation between BMI and levels of gene expression of leptin and 11βHSD1. Conclusion: Gene expression of leptin, PPARγ1, and 11βHSD1 in AT is modified in human obesity. This default is completely corrected by RYGB. Copyright © 2012 S. Karger GmbH, Freiburg.

Significant molecular and systemic adaptations after repeated sprint training in hypoxia

While intermittent hypoxic training (IHT) has been reported to evoke cellular responses via hypoxia inducible factors (HIFs) but without substantial performance benefits in endurance athletes, we hypothesized that repeated sprint training in hypoxia could enhance repeated sprint ability (RSA) performed in normoxia via improved glycolysis and O(2) utilization. 40 trained subjects completed 8 cycling repeated sprint sessions in hypoxia (RSH, 3000 m) or normoxia (RSN, 485 m). Before (Pre-) and after (Post-) training, muscular levels of selected mRNAs were analyzed from resting muscle biopsies and RSA tested until exhaustion (10-s sprint, work-to-rest ratio 1ratio2) with muscle perfusion assessed by near-infrared spectroscopy. From Pre- to Post-, the average power output of all sprints in RSA was increased (p<0.01) to the same extent (6% vs 7%, NS) in RSH and in RSN but the number of sprints to exhaustion was increased in RSH (9.4+/-4.8 vs. 13.0+/-6.2 sprints, p<0.01) but not in RSN (9.3+/-4.2 vs. 8.9+/-3.5). mRNA concentrations of HIF-1alpha (+55%), carbonic anhydrase III (+35%) and monocarboxylate transporter-4 (+20%) were augmented (p<0.05) whereas mitochondrial transcription factor A (-40%), peroxisome proliferator-activated receptor gamma coactivator 1alpha (-23%) and monocarboxylate transporter-1 (-36%) were decreased (p<0.01) in RSH only. Besides, the changes in total hemoglobin variations (Delta[tHb]) during sprints throughout RSA test increased to a greater extent (p<0.01) in RSH. Our findings show larger improvement in repeated sprint performance in RSH than in RSN with significant molecular adaptations and larger blood perfusion variations in active muscles.

Anti-vascular endothelial growth factor acts on retinal microglia/macrophage activation in a rat model of ocular inflammation.

PURPOSE: To evaluate whether anti-vascular endothelial growth factor (VEGF) neutralizing antibodies injected in the vitreous of rat eyes influence retinal microglia and macrophage activation. To dissociate the effect of anti-VEGF on microglia and macrophages subsequent to its antiangiogenic effect, we chose a model of acute intraocular inflammation. METHODS: Lewis rats were challenged with systemic lipopolysaccharide (LPS) injection and concomitantly received 5 µl of rat anti-VEGF-neutralizing antibody (1.5 mg/ml) in the vitreous. Rat immunoglobulin G (IgG) isotype was used as the control. The effect of anti-VEGF was evaluated at 24 and 48 h clinically (uveitis scores), biologically (cytokine multiplex analysis in ocular media), and histologically (inflammatory cell counts on eye sections). Microglia and macrophages were immunodetected with ionized calcium-binding adaptor molecule 1 (IBA1) staining and counted based on their differential shapes (round amoeboid or ramified dendritiform) on sections and flatmounted retinas using confocal imaging and automatic quantification. Activation of microglia was also evaluated with inducible nitric oxide synthase (iNOS) and IBA1 coimmunostaining. Coimmunolocalization of VEGF receptor 1 and 2 (VEGF-R1 and R2) with IBA1 was performed on eye sections with or without anti-VEGF treatment. RESULTS: Neutralizing rat anti-VEGF antibodies significantly decreased ocular VEGF levels but did not decrease the endotoxin-induced uveitis (EIU) clinical score or the number of infiltrating cells and cytokines in ocular media (interleukin [IL]-1β, IL-6, tumor necrosis factor [TNF]-α, and monocyte chemoattractant protein [MCP]-1). Eyes treated with anti-VEGF showed a significantly decreased number of activated microglia and macrophages in the retina and the choroid and decreased iNOS-positive microglia. IBA1-positive cells expressed VEGF-R1 and R2 in the inflamed retina. CONCLUSIONS: Microglia and macrophages expressed VEGF receptors, and intravitreous anti-VEGF influenced the microglia and macrophage activation state. Taking into account that anti-VEGF drugs are repeatedly injected in the vitreous of patients with retinal diseases, part of their effects could result from unsuspected modulation of the microglia activation state. This should be further studied in other ocular pathogenic conditions and human pathology.

Estrogenic activity assessment of environmental chemicals using in vitro assays: identification of two new estrogenic compounds.

Environmental chemicals with estrogenic activities have been suggested to be associated with deleterious effects in animals and humans. To characterize estrogenic chemicals and their mechanisms of action, we established in vitro and cell culture assays that detect human estrogen receptor [alpha] (hER[alpha])-mediated estrogenicity. First, we assayed chemicals to determine their ability to modulate direct interaction between the hER[alpha] and the steroid receptor coactivator-1 (SRC-1) and in a competition binding assay to displace 17ss-estradiol (E(2)). Second, we tested the chemicals for estrogen-associated transcriptional activity in the yeast estrogen screen and in the estrogen-responsive MCF-7 human breast cancer cell line. The chemicals investigated in this study were o,p'-DDT (racemic mixture and enantiomers), nonylphenol mixture (NPm), and two poorly analyzed compounds in the environment, namely, tris-4-(chlorophenyl)methane (Tris-H) and tris-4-(chlorophenyl)methanol (Tris-OH). In both yeast and MCF-7 cells, we determined estrogenic activity via the estrogen receptor (ER) for o,p'-DDT, NPm, and for the very first time, Tris-H and Tris-OH. However, unlike estrogens, none of these xenobiotics seemed to be able to induce ER/SRC-1 interactions, most likely because the conformation of the activated receptor would not allow direct contacts with this coactivator. However, these compounds were able to inhibit [(3)H]-E(2) binding to hER, which reveals a direct interaction with the receptor. In conclusion, the test compounds are estrogen mimics, but their molecular mechanism of action appears to be different from that of the natural hormone as revealed by the receptor/coactivator interaction analysis.

PlGF-1 and VEGFR-1 pathway regulation of the external epithelial hemato-ocular barrier. A model for retinal edema.

VEGF is considered as an important factor in the pathogenesis of macular edema. VEGF induces the rupture of the blood retinal barrier and may also influence the retinal pigment epithelial (RPE) outer retinal barrier. The aim of this work was to analyze the influence of the VEGF receptor pathways in the modulation of the RPE barrier breakdown in vitro and in vivo. The ARPE19 human junctions in culture are modulated by VEGF through VEGFR-1 but not through VEGFR-2. PlGF-1, that is a pure agonist of VEGFR-1, is produced in ARPE-19 cells under hypoxic conditions and mimics VEGF effects on the external retinal barrier as measured by TER and inulin flux. In vivo, the intravitreous injection of PlGF-1 induces a rupture of the external retinal barrier together with a retinal edema. This effect is reversible within 4 days. VEGF-E, that is a pure agonist of VEGFR-2, does not induce any acute effect on the RPE barrier. These results demonstrate that PlGF-1 can reproduce alterations of the RPE barrier occurring during diabetic retinopathy.

Hepatic deficiency in transcriptional cofactor TBL1 promotes liver steatosis and hypertriglyceridemia.

The aberrant accumulation of lipids in the liver ("fatty liver") is tightly associated with several components of the metabolic syndrome, including type 2 diabetes, coronary heart disease, and atherosclerosis. Here we show that the impaired hepatic expression of transcriptional cofactor transducin beta-like (TBL) 1 represents a common feature of mono- and multigenic fatty liver mouse models. Indeed, the liver-specific ablation of TBL1 gene expression in healthy mice promoted hypertriglyceridemia and hepatic steatosis under both normal and high-fat dietary conditions. TBL1 deficiency resulted in inhibition of fatty acid oxidation due to impaired functional cooperation with its heterodimerization partner TBL-related (TBLR) 1 and the nuclear receptor peroxisome proliferator-activated receptor (PPAR) α. As TBL1 expression levels were found to also inversely correlate with liver fat content in human patients, the lack of hepatic TBL1/TBLR1 cofactor activity may represent a molecular rationale for hepatic steatosis in subjects with obesity and the metabolic syndrome.

Cellular distribution of glucose and monocarboxylate transporters in human brain white matter and multiple sclerosis lesions.

To ensure efficient energy supply to the high demanding brain, nutrients are transported into brain cells via specific glucose (GLUT) and monocarboxylate transporters (MCT). Mitochondrial dysfunction and altered glucose metabolism are thought to play an important role in the progression of neurodegenerative diseases, including multiple sclerosis (MS). Here, we investigated the cellular localization of key GLUT and MCT proteins in human brain tissue of non-neurological controls and MS patients. We show that in control brain tissue GLUT and MCT proteins were abundantly expressed in a variety of central nervous system cells, particularly in microglia and endothelial cells. In active MS lesions, GLUTs and MCTs were highly expressed in infiltrating leukocytes and reactive astrocytes. Astrocytes manifest increased MCT1 staining and maintain GLUT expression in inactive lesions, whereas demyelinated axons exhibit significantly reduced GLUT3 and MCT2 immunoreactivity in inactive lesions. Finally, we demonstrated that the co-transcription factor peroxisome proliferator-activated receptor gamma co-activator 1-alpha (PGC-1α), an important protein involved in energy metabolism, is highly expressed in reactive astrocytes in active MS lesions. Overexpression of PGC-1α in astrocyte-like cells resulted in increased production of several GLUT and MCT proteins. In conclusion, we provide for the first time a comprehensive overview of key nutrient transporters in white matter brain samples. Moreover, our data demonstrate an altered expression of these nutrient transporters in MS brain tissue, including a marked reduction of axonal GLUT3 and MCT2 expression in chronic lesions, which may impede efficient nutrient supply to the hypoxic demyelinated axons thereby contributing to the ongoing neurodegeneration in MS. GLIA 2014;62:1125-1141.

Fate of TLR-1/TLR-2 agonist functionalised pDNA nanoparticles upon deposition at the human bronchial epithelium in vitro.

BACKGROUND: Plasmid DNA vaccination is a promising approach, but studies in non-human primates and humans failed to achieve protective immunity. To optimise this technology further with focus on pulmonary administration, we developed and evaluated an adjuvant-equipped DNA carrier system based on the biopolymer chitosan. In more detail, the uptake and accompanying immune response of adjuvant Pam3Cys (Toll-like receptor-1/2 agonist) decorated chitosan DNA nanoparticles (NP) were explored by using a three-dimensional (3D) cell culture model of the human epithelial barrier. Pam3Cys functionalised and non-functionalised chitosan DNA NP were sprayed by a microsprayer onto the surface of 3D cell cultures and uptake of NP by epithelial and immune cells (blood monocyte-derived dendritic cells (MDDC) and macrophages (MDM)) was visualised by confocal laser scanning microscopy. In addition, immune activation by TLR pathway was monitored by analysis of interleukin-8 and tumor necrosis factor-α secretions (ELISA). RESULTS: At first, a high uptake rate into antigen-presenting cells (MDDC: 16-17%; MDM: 68-75%) was obtained. Although no significant difference in uptake patterns was observed for Pam3Cys adjuvant functionalised and non-functionalised DNA NP, ELISA of interleukin-8 and tumor necrosis factor-α demonstrated clearly that Pam3Cys functionalisation elicited an overall higher immune response with the ranking of Pam3Cys chitosan DNA NPâeuro0/00>âeuro0/00chitosan DNA NPâeuro0/00=âeuro0/00DNA unloaded chitosan NPâeuro0/00>âeuro0/00control (culture medium). CONCLUSIONS: Chitosan-based DNA delivery enables uptake into abluminal MDDC, which are the most immune competent cells in the human lung for the induction of antigen-specific immunity. In addition, Pam3Cys adjuvant functionalisation of chitosan DNA NP enhances significantly an environment favoring recruitment of immune cells together with a Th1 associated (cellular) immune response due to elevated IL-8 and TNF-α levels. The latter renders this DNA delivery approach attractive for potential DNA vaccination against intracellular pathogens in the lung (e.g., Mycobacterium tuberculosis or influenza virus).

Absence of intestinal PPARγ aggravates acute infectious colitis in mice through a lipocalin-2-dependent pathway.

To be able to colonize its host, invading Salmonella enterica serovar Typhimurium must disrupt and severely affect host-microbiome homeostasis. Here we report that S. Typhimurium induces acute infectious colitis by inhibiting peroxisome proliferator-activated receptor gamma (PPARγ) expression in intestinal epithelial cells. Interestingly, this PPARγ down-regulation by S. Typhimurium is independent of TLR-4 signaling but triggers a marked elevation of host innate immune response genes, including that encoding the antimicrobial peptide lipocalin-2 (Lcn2). Accumulation of Lcn2 stabilizes the metalloproteinase MMP-9 via extracellular binding, which further aggravates the colitis. Remarkably, when exposed to S. Typhimurium, Lcn2-null mice exhibited a drastic reduction of the colitis and remained protected even at later stages of infection. Our data suggest a mechanism in which S. Typhimurium hijacks the control of host immune response genes such as those encoding PPARγ and Lcn2 to acquire residence in a host, which by evolution has established a symbiotic relation with its microbiome community to prevent pathogen invasion.

Transmission of HIV-1 drug resistance in Switzerland: a 10-year molecular epidemiology survey.

OBJECTIVES: Representative prevalence data of transmitted drug-resistant HIV-1 are essential to establish accurate guidelines addressing resistance testing and first-line treatments. METHODS: Systematic resistance testing was carried out in individuals in Switzerland with documented HIV-1 seroconversion during 1996-2005 and available samples with RNA > 1000 copies/ml obtained within 1 year of estimated seroconversion. Resistance interpretation used the Stanford list of mutations for surveillance of transmitted drug resistance and the French National Agency for AIDS Research algorithm. RESULTS: Viral sequences from 822 individuals were available. Risk groups were men having sex with men (42%), heterosexual contacts (32%) and intravenous drug users (20%); 30% were infected with non-B subtype viruses. Overall, prevalence of transmitted resistance was 7.7% [95% confidence interval (CI), 5.9-9.5] for any drug, 5.5% (95% CI, 3.9-7.1) for nucleoside reverse transcriptase inhibitors, 1.9% (95% CI, 1.0-2.8) for non-nucleoside reverse transcriptase inhibitors and 2.7% (95% CI, 1.6-3.8) for protease inhibitors. Dual- or triple-class resistance was observed in 2% (95% CI, 0.8-2.5). No significant trend in prevalence of transmitted resistance was observed over years. There were no differences according to ethnicity, risk groups or gender, but prevalence of transmitted resistance was highest among individuals infected with subtype B virus. CONCLUSIONS: The transmission rate of drug-resistant HIV-1 has been stable since 1996, with very rare transmission of dual- or triple-class resistance. These data suggest that transmission of drug resistance in the setting of easy access to antiretroviral treatment can remain stable and be kept at a low level.

Type I interferons protect T cells against NK cell attack mediated by the activating receptor NCR1.

Direct type I interferon (IFN) signaling on T cells is necessary for the proper expansion, differentiation, and survival of responding T cells following infection with viruses prominently inducing type I IFN. The reasons for the abortive response of T cells lacking the type I IFN receptor (Ifnar1(-/-)) remain unclear. We report here that Ifnar1(-/-) T cells were highly susceptible to natural killer (NK) cell-mediated killing in a perforin-dependent manner. Depletion of NK cells prior to lymphocytic choriomeningitis virus (LCMV) infection completely restored the early expansion of Ifnar1(-/-) T cells. Ifnar1(-/-) T cells had elevated expression of natural cytotoxicity triggering receptor 1 (NCR1) ligands upon infection, rendering them targets for NCR1 mediated NK cell attack. Thus, direct sensing of type I IFNs by T cells protects them from NK cell killing by regulating the expression of NCR1 ligands, thereby revealing a mechanism by which T cells can evade the potent cytotoxic activity of NK cells.

PPARγ Agonist-Induced Fluid Retention Depends on αENaC Expression in Connecting Tubules.

BACKGROUND/AIMS: Thiazolidinediones (TZDs, like rosiglitazone (RGZ)) are peroxisome proliferator-activated receptor γ (PPARγ) agonists used to treat type 2 diabetes. Clinical limitations include TZD-induced fluid retention and body weight (BW) increase, which are inhibited by amiloride, an epithelial-sodium channel (ENaC) blocker. RGZ-induced fluid retention is maintained in mice with αENaC knockdown in the collecting duct (CD). Since ENaC in the connecting tubule (CNT) rather than in CD appears to be critical for normal NaCl retention, we aimed to further explore the role of ENaC in CNT in RGZ-induced fluid retention. METHODS: Mice with conditional inactivation of αENaC in both CNT and CD were used (αENaC lox/lox AQP2-Cre; 'αENaC-CNT/CD-KO') and compared with littermate controls (αENaC lox/lox mice; 'WT'). BW was monitored and total body water (TBW) and extracellular fluid volume (ECF) were determined by bioelectrical impedance spectroscopy (BIS) before and after RGZ (320 mg/kg diet for 10 days). RESULTS: On regular NaCl diet, αENaC-CNT/CD-KO had normal BW, TBW, ECF, hematocrit, and plasma Na(+), K(+), and creatinine, associated with an increase in plasma aldosterone compared with WT. Challenging αENaC-CNT/CD-KO with a low NaCl diet unmasked impaired NaCl and K homeostasis, consistent with effective knockdown of αENaC. In WT, RGZ increased BW (+6.1%), TBW (+8.4%) and ECF (+10%), consistent with fluid retention. These changes were significantly attenuated in αENaC-CNT/CD-KO (+3.4, 1.3, and 4.3%). CONCLUSION: Together with the previous studies, the current results are consistent with a role of αENaC in CNT in RGZ-induced fluid retention, which dovetails with the physiological relevance of ENaC in this segment. © 2014 S. Karger AG, Basel.